Abstract:

The invention relates to aqueous aerosol preparations for inhalation,
containing therapeutically active micro-organisms or parts of
micro-organisms as the active ingredient.

Claims:

1. An aqueous aerosol preparation for inhalation comprising
micro-organisms or parts of micro-organisms as active substance,
characterized in that the aqueous aerosol preparation comprises the
active substance in a therapeutically effective form.

2. The aqueous aerosol preparation according to claim 1, characterized in
that the aqueous aerosol preparation comprises the active substance in a
concentration of between 3 mg/ml and 100 mg/ml.

3. The aqueous aerosol preparation according to claim 1, characterized in
that micro-organisms of the genus Bacillus, Staphylococcus, Pseudomonas,
Escherichia, Salmonella, or a mixture of these genera are the active
substance.

4. The aqueous aerosol preparation according to claim 1, characterized in
that micro-organisms of the species Bacillus subtilis, Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella abony or a
mixture of these species are the active substance.

5. The aqueous aerosol preparation according to claim 1, characterized in
that the preparation comprises one or more adjuvants selected from among
the surfactants, emulsifiers, stabilisers, permeation enhancers, and
preservatives, and combinations thereof.

6. The aqueous aerosol preparation according to claim 1, characterized in
that the preparation also comprises an amino acid for improving the
solubility/stability of the active substance.

7. The aqueous aerosol preparation according to claim 6, characterized in
that the preparation is suitable for use in a propellant-free nebulizer.

8. The aqueous aerosol preparation according to claim 7, characterized in
that the preparation has a limiting viscosity of up to
1600.times.10.sup.-6 Pascal.

9-15. (canceled)

16. A propellant-free nebulizer with an aqueous aerosol preparation for
inhalation which aqueous aerosol preparation comprises micro-organisms or
parts of micro-organisms as active substance, characterized in that a
single dose of the aqueous aerosol preparation is measured in a measuring
chamber and is sprayed at high pressure of between 100 and 500 bar
through at least one nozzle with a hydraulic diameter of 1 to 12 microns
to form inhalable droplets with a particle size of less than 10 microns
within a time of between one and two seconds.

17. The propellant-free nebulizer of claim 16 characterized in that the
single dose is between 10 and 20 microliters.

18. The propellant-free nebulizer of claim 16 characterized in that the
nebulizer has two nozzles that are directed so that the two jets meet in
such a way that the aqueous aerosol preparation is nebulized.

Description:

[0002]The use of medicaments in the form of inhalable aerosols has long
been known. Such aerosols are used not only for the treatment of
respiratory complaints such as asthma; they are also used when the lungs
or nasal mucosa are to be the organ of absorption. Frequently, the blood
levels obtained with the active substances are high enough to treat
diseases in other parts of the body. Inhalable aerosols may also be used
as vaccines.

[0003]A number of methods are used in practice to prepare aerosols. Either
suspensions or solutions of active substances are sprayed, using
propellant gases inter alia, or active substances in the form of
micronised powders are subjected to a vortex in the air breathed in or
finally aqueous solutions are atomised using atomisers.

[0004]In molecules of a more complex structure, such as interferons, for
example, the atomisation of aqueous solutions may easily lead to an
undesirable reduction in the activity of the active substance, presumably
as a result of shear forces and heating. It is suspected that the
formation of less active protein aggregates plays a part in this process.
In their article "Stability of recombinant consensus interferon to
air-jet and ultrasonic atomisation", J. Pharm. Sci. 84:1210-1214 [1995],
A. Y. Ip and colleagues described examples of the formation of interferon
aggregates after ultrasound or nozzle spraying with a concomitant loss of
the biological activity of the interferon. Even if the destruction of the
biomolecule is not complete, the reduction in activity described here is
important as it causes greater consumption of the possibly expensive
biomolecule and allows the dosage of active medicament per spray to
become inaccurate. This reduction in the activity of molecules of a
complicated structure during the aerosol production is not limited to
interferons but also occurs to a greater or lesser extent during the
aerosol spraying of other proteins (cf. e.g. Niven et al, Pharm Res. 12:
53-59 [1995]) and biomolecules, particularly macromolecules of this kind.

[0005]It is also known to treat mucoviscidosis patients with sulphide
bridge cleaving enzymes by atomising these enzymes.

[0007]Besides the industrial production of the aerosol containing the
biomolecule, a second step is needed to ensure that the biomolecules are
absorbed in the lungs. The lung in an adult human has a large absorption
surface but also has a number of obstacles to the pulmonary absorption of
biomolecules. After inspiration through the nose or mouth, air (with the
aerosol that it carries) passes into the trachea and then through
progressively smaller bronchi and bronchioles into the alveoli. The
alveoli have a much large surface area than the trachea, bronchi and
bronchioles put together. They are the main absorption zone, not only for
oxygen but also for biologically active macromolecules. In order to pass
from the air into the bloodstream, molecules have to cross the alveolar
epithelium, the capillary endothelium and the lymph-containing
interstitial space between these two layers of cells. This may take place
by active or passive transport processes. The cells in these two layers
of cells are close together, so that most of the large biological
macromolecules (such as proteins, for example) cross this barrier much
more slowly than smaller molecules. The process of crossing the alveolar
epithelium and the capillary endothelium competes with other biological
processes that lead to the destruction of the biomolecule. The
bronchoalveolar liquid contains exoproteases [cf. e.g. Wall D. A. and
Lanutti, A. T. `High levels of exopeptidase activity are present in rat
and canine bronchoalveolar lavage fluid`. Int. J. Pharm. 97:171-181
(1993)]. It also contains macrophages which eliminate inhaled protein
particles by phagocytosis. These macrophages migrate to the base of the
bronchial tree, from where they are expelled from the lungs by the
mucociliary clearance mechanism. They may then migrate into the lymphatic
system. Moreover, the macrophages may be influenced by the aerosolised
protein in their physiology, e.g. interferons may activate alveolar
macrophages. The migration of activated macrophages is a further
mechanism for propagating the systemic effect of an inhaled protein. The
complexity of this process means that results of aerosol tests with one
type of protein can only be transferred to another type of protein to a
limited extent. Small differences between interferons may for example
have a significant influence on their susceptibility to the degradation
mechanisms in the lungs [cf. Bocci et al `Pulmonary catabolism of
interferons: alveolar absorption of 125-I labelled human interferon
alpha is accompanied by partial loss of biological activity` Antiviral
Research 4:211-220 (1984)].

[0008]Micro-organisms, which are a form of biological macromolecules, may
indeed be atomised in principle, but these atomisation generally takes
place with a loss of activity. The definition of micro-organisms in this
context encompasses all tiny single-cell living organisms with a size of
≦200 μm. Micro-organisms include in particular bacteria and
fungi.

[0009]The aim of the present invention is to provide aqueous aerosol
preparations which contain therapeutically effective micro-organisms or
parts of micro-organisms, particularly bacteria, fungi or parts thereof,
as active substance and can be used for inhalation.

[0010]Surprisingly it has been found that liquid preparations of
therapeutically active micro-organisms or parts of micro-organisms as
active substance can be atomised without any appreciable loss of
activity.

[0011]Preferably the atomisers used are propellant-free atomisers which
spray a predetermined amount of an aerosol preparation at high pressure
between 100 and 500 bar through at least one nozzle with a hydraulic
diameter of 1 to 12 microns, so as to obtain inhalable droplets with an
average particle size of less than 10 microns.

[0012]Moreover, highly concentrated solutions of therapeutically effective
micro-organisms or parts of micro-organisms may also be atomised. The use
of highly concentrated solutions makes it possible to use a device with a
number of single doses in a small reservoir.

[0013]The invention also relates to aerosol preparations in the form of
aqueous solutions which contain, as active substance, therapeutically
effective micro-organisms or parts of micro-organisms, particularly
therapeutically effective bacteria, fungi or parts of bacteria or fungi,
in an amount of between 3 mg/ml and 100 mg/ml. Particularly preferred are
the micro-organisms of the Bacillus, Staphylococcus, Pseudomonas,
Escherichia, Salmonella, Candida or Aspergillus strains or a mixture of
these strains. Most particularly preferred are micro-organisms of the
types Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa,
Escherichia coli, Salmonella abony, Candida albicans, Aspergillus niger,
or a mixture of these types.

[0014]Surprisingly it has been found that more viscous solutions of
therapeutically active micro-organisms or parts of micro-organisms may be
sprayed to form inhalable droplets of a suitable droplet size.

[0015]This allows larger amounts of active substance to be administered in
each dose and thus increases the therapeutic efficacy of therapeutically
active micro-organisms or parts of micro-organisms in inhalative therapy.

[0016]For the aerosol according to the invention, therapeutically
effective micro-organisms or parts of micro-organisms containing aqueous
aerosol preparations up to a limiting viscosity of 1600×10-6
Pascal are used.

[0017]More highly viscous solutions of therapeutically active
micro-organisms or parts of micro-organisms, having a limiting viscosity
of up to 1100×10-6 Pascal, are preferred. The limiting
viscosities stated were determined using an Oswald viscosimeter by a
method known from the literature. As a comparison: the limiting viscosity
of water is 900×10-6 Pascal.

[0018]The aqueous aerosol preparation may also contain one or more
adjuvants selected from among the surfactants, emulsifiers, stabilisers,
permeation promoters and/or preservatives as well as an amino acid to
improve the solubility/stability of the active substance, preferably
proline.

[0019]The invention also relates to the use of the aqueous aerosol
preparation for the treatment of respiratory complaints, particularly for
the treatment of chronic obstructive pulmonary disease (COPD) or for the
immune treatment of humans and animals.

[0020]The atomiser used may be any of the conventional devices, with or
without propellant gas.

[0021]A new generation of propellant-free atomisers is described in U.S.
Pat. No. 5,497,944 and WO 97/12687, the contents of which are hereby
incorporated by reference. A preferred nozzle arrangement for nebulising
the aqueous aerosol preparations of biologically active macromolecules
according to the invention is shown in FIG. 8 of the U.S. patent. The
particular advantage of the nebulisers described therein is that no
propellant gases are used.

[0022]A further developed embodiment of the atomisers described therein is
disclosed in PCT/EP96/04351=WO 97/12687. In relation to the present
invention reference is made expressly to FIG. 6 described therein and the
associated parts of the description of the application. The nebuliser
described therein may advantageously be used to produce the claimed
inhalable aerosols of biologically active macromolecules. In the
nebulisers described therein, active substance-containing solutions of
defined volumes are sprayed through small nozzles at high pressures, so
as to obtain inhalable aerosols with an average particle size of between
3 and 10 microns.

[0023]Of particular importance is the use of the device described in the
above-mentioned patent or patent application for propellant-free
atomisation of the aerosol preparation according to the invention. The
atomiser (nebuliser) essentially consists of the upper housing part, a
pump housing, a nozzle, a locking clamp, a spring housing, a spring and a
storage container, characterised by [0024]a pump housing fixed in the
upper housing part and carrying at one end a nozzle body with the nozzle,
[0025]a hollow piston with valve body, [0026]a power take-off flange in
which the hollow body is fixed and which is located in the upper housing
part, [0027]a locking clamping mechanism located in the upper housing
part, [0028]a spring housing with the spring located therein, which is
rotatably mounted on the upper housing part by means of a rotary bearing,
[0029]a lower housing part which is fitted onto the spring housing in the
axial direction.

[0030]The hollow piston with valve body corresponds one of the
above-mentioned devices. It projects partially into the cylinder of the
pump housing and is disposed to be axially movable in the cylinder. At
the moment of release of the spring the hollow piston with valve body
exerts, at its high pressure end, a pressure of 5 to 60 Mpa (about 50 to
600 bar), preferably 10 to 60 Mpa (about 100 to 600 bar) on the fluid.

[0031]The nozzle in the nozzle body is preferably microstructured, i.e.
manufactured by micro-engineering. Microstructured nozzle bodies are
disclosed for example in WO-94/07607; reference is hereby made to the
contents of this specification.

[0032]The nozzle body consists for example of two sheets of glass and/or
silicon securely fixed together, at least one of which has one or more
microstructured channels which connect the nozzle inlet end to the nozzle
outlet end. At the nozzle outlet end there is at least one round or
non-round opening less than or equal to 10 μm.

[0033]The directions of spraying of the nozzles in the nozzle body may run
parallel to each other or may be inclined relative to one another. In the
case of a nozzle body having at least two nozzle openings at the outlet
end, the directions of spraying may be inclined relative to one another
at an angle of 20 degrees to 160 degrees, preferably at an angle of 60 to
150 degrees.

[0034]The directions of spraying meet in the region of the nozzle
openings.

[0035]The valve body is preferably mounted at the end of the hollow piston
which faces the nozzle body.

[0036]The locking clamping mechanism contains a spring, preferably a
cylindrical helical compression spring as a store for the mechanical
energy. The spring acts on the power take-off flange as a spring member
the movement of which is determined by the position of a locking member.
The travel of the power take-off flange is precisely limited by an upper
stop and a lower stop. The spring is preferably tensioned via a
stepping-up gear, e.g. a helical sliding gear, by an external torque
which is generated when the upper housing part is turned relative to the
spring housing in the lower housing part. In this case, the upper housing
part and the power take-off flange contain a single- or multi-speed
spline gear.

[0037]The locking member with engaging locking surfaces is arranged in an
annular configuration around the power take-off flange. It consists for
example of a ring of plastics or metal which is inherently radially
elastically deformable. The ring is arranged in a plane perpendicular to
the axis of the atomiser. After the locking of the spring, the locking
surfaces of the locking member slide into the path of the power take-off
flange and prevent the spring from being released. The locking member is
actuated by means of a button. The actuating button is connected or
coupled to the locking member. In order to actuate the locking clamping
mechanism the actuating button is moved parallel to the annular plane,
preferably into the atomiser, and the deformable ring is thereby deformed
in the annular plane.

[0038]The lower housing part is pushed axially over the spring housing and
covers the bearing, the drive for the spindle and the storage container
for the fluid.

[0039]When the atomiser is operated, the upper part of the housing is
rotated relative to the lower part, the lower part taking the spring
housing with it. The spring meanwhile is compressed and biased by means
of the helical sliding gear, and the clamping mechanism engages
automatically. The angle of rotation is preferably a whole-number
fraction of 360 degrees, e.g. 180 degrees. At the same time as the spring
is tensioned, the power take-off component in the upper housing part is
moved along by a given amount, the hollow piston is pulled back inside
the cylinder in the pump housing, as a result of which some of the fluid
from the storage container is sucked into the high pressure chamber in
front of the nozzle.

[0040]If desired, a plurality of replaceable storage containers containing
the fluid to be atomised can be inserted in the atomiser one after
another and then used. The storage container contains the aqueous aerosol
preparation according to the invention.

[0041]The effectiveness of a nebulisation device can be tested in an in
vitro system, by nebulising a protein solution and catching and analysing
the aerosol. The activity of the protein in the nebulisation solution (a)
is compared with the activity in the analysed aerosol (b), e.g. by means
of an immunoassay or an assay of the biological activity of the protein.
This experiment makes it possible to evaluate the degree of destruction
of the protein by the nebulisation process.

[0042]A second parameter for evaluating the aerosol quality is the
so-called inhalable fraction which is defined here as the proportion of
droplets of mist with a mass median aerodynamic diameter (MMAD) of less
than 5.8 μm. The MMAD may be measured e.g. using an "Andersen Cascade
Impactor". For efficient protein absorption it is important not only to
achieve nebulisation with no appreciable loss of activity but also to
generate an aerosol with a good (approx. 60%) inhalable fraction.
Aerosols with an MMAD of less than 5.8 μm are significantly more
suitable for reaching the alveoli, their chances of being absorbed being
clearly greater on account of the very great absorbent surface. The
effectiveness of a nebulising device can also be tested in an in vivo
system, in which case factors such as susceptibility to lung proteases
come into play. As an example of an in vivo test system, a
protein-containing mist may be administered to a dog through a tracheal
tube. Blood samples are taken at suitable intervals and then the protein
level in the plasma is measured using immunological or biological
methods.

[0043]The following in vivo tests are described to illustrate advantages
of the aerosol according to the invention.

[0052]A number of sprays corresponding to a total volume of approx. 0.5 ml
of were released using the Respimat®. The aerosol produced was
captured in a sealed 1000 ml shaking flask with 100 ml of a physiological
buffer solution and 0.1% Tween 80. Then the flask was sealed off
completely at its opening and the aerosol was taken up in the buffer
solution in the flask by gentle shaking.

[0053]The amount of aerosol released was determined by weighing the
Respimat® inhaler. The subsequent microbiological tests were carried
out according to the instructions in Ph. Eur. 3, 2000 (2.6.12) and USP
24:

[0054]20 ml of the buffer solution from the shaking flask and one aliquot
of 0.1 ml of the [noun omitted] in the reservoir of the Respimat®
inhaler were filtered through membrane filters separately from one
another. As a control batch, 0.1 ml of the corresponding suspension of
the above-mentioned micro-organisms in 20 ml buffer solution are
filtered.

[0055]The membrane filters through which the suspensions of bacteria were
filtered were placed on agar plates after the filtration and incubated
for 5 days at 33° C.

[0056]The membrane filters through which the suspensions of yeasts and
fungi were filtered were placed on agar plates after the filtration and
incubated for 5 days at 25° C. In all, three tests are carried out
on each micro-organism. Tab. 1, Tab. 3 and Tab. 5 show the results of the
three tests. As a comparison, the colony-forming units per millilitre
(CFU/ml) from the aerosol, the reservoir and the control group are shown.
The survival rate and death rate in percent were calculated for the
captured aerosol in relation to the reservoir suspension.

[0057]Tab. 2, Tab. 4 and Tab. 6 show the results for the amount of aerosol
released, determined by weighing.

[0058]Tab. 7 shows the statistical data of the survival rate. More than
87% surviving micro-organisms were found for Bacillus subtilis,
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and
Salmonella abony. This means that between 87 and 95 percent of the
micro-organisms that were nebulised were still capable of dividing and
growing after the nebulisation and working up of the aerosol. This is an
indication that the micro-organisms have survived the nebulisation.

[0059]The rates of Candida albicans and Aspergillus niger are below 3%.
Tab. 8 shows the corresponding death rate. This means that between 97 and
100 percent of the micro-organisms that were nebulised were no longer
capable of dividing and growing after the nebulisation and working up of
the aerosol. This is an indication that the micro-organisms have either
not survived the nebulisation or because of their size have been retained
by the filter mechanisms in the Respimat® inhaler.

[0060]The results of the tests described above show that generally after
being used and nebulised in the Respimat® inhaler bacteria show no
loss of activity.

[0061]Very large micro-organisms such as e.g. yeasts and fungi (Candida
albicans, Aspergillus niger) are obviously retained in the Respimat
because of their size. They cannot pass through the filters of the
Respimat® inhaler.

[0062]On account of the high variability in biological tests, it can be
assumed that when nebulised in the Respimat® inhaler bacteria are not
killed off in practice or held back by filtration. The conversion of
bacterial suspensions into aerosols in the Respimat® inhaler has no
effect on the vitality of the micro-organisms. Thus, bacteria or
components of bacteria can be efficiently transported into the human lung
for curative purposes.